Rho-kinase/myosin light chain kinase pathway plays a key role in the impairment of bile canaliculi dynamics induced by cholestatic drugs

Abstract

Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injury; however, the mechanisms underlying such injuries are poorly understood. In this study of human HepaRG and primary hepatocytes, we found that bile canaliculi (BC) underwent spontaneous contractions, which are essential for bile acid (BA) efflux and require alternations in myosin light chain (MLC2) phosphorylation/dephosphorylation. Short exposure to 6 cholestatic compounds revealed that BC constriction and dilation were associated with disruptions in the ROCK/MLCK/myosin pathway. At the studied concentrations, cyclosporine A and chlorpromazine induced early ROCK activity, resulting in permanent MLC2 phosphorylation and BC constriction. However, fasudil reduced ROCK activity and caused rapid, substantial and permanent MLC2 dephosphorylation, leading to BC dilation. The remaining compounds (1-naphthyl isothiocyanate, deoxycholic acid and bosentan) caused BC dilation without modulating ROCK activity, although they were associated with a steady decrease in MLC2 phosphorylation via MLCK. These changes were associated with a common loss of BC contractions and failure of BA clearance. These results provide the first demonstration that cholestatic drugs alter BC dynamics by targeting the ROCK/MLCK pathway; in addition, they highlight new insights into the mechanisms underlying bile flow failure and can be used to identify new predictive biomarkers of drug-induced cholestasis.

WITHDRAWN: Macrophage-targeting Fasudil treatment protects liver from the ischemia/reperfusion injury by promoting M2 macrophage polarization

“…Fasudil is a potent Rho-associated protein kinase (ROCK) inhibitor [8,9,10,11,12,13] and vasodilator and has been used for the treatment of cerebral vasospasm [9] and many other diseases, such as cancer [14,15], allergy [16] and inflammation [17,18]. The systematic administration of Fasudil was reported protecting mice from experimental autoimmune encephalomyelitis (EAE) and shifting macrophages from M1 to M2 [19].…”

Differential sensitivity of metabolically competent and non-competent HepaRG cells to apoptosis induced by diclofenac combined or not with TNF-α

“…As observed with several other cholestatic drugs (Sharanek et al, 2016), we found that DCF provoked early dilatation of bile canaliculi at lower concentrations than those causing cytotoxicity and these bile canaliculi deformations were associated with alterations of taurocholic acid clearance as well as of expression of influx and efflux bile acid transporters. Although TNF-α did not obviously alter these major DCF cholestatic effects when added prior to and/or simultaneously with the drug this did not exclude that this cytokine could modulate bile acids synthesis and/or secretion.…”

The ascending pathophysiology of cholestatic liver disease

“…Downstream effects of these knockouts range from reduced phosphorylation of adenosine monophosphate-activated protein kinase (30) to disruption of tight-junctional proteins such as Claudin-1 and zona occludens 1 (31) to reduced Wnt signaling activity. (32) The cholestasisinducing drug fasudil inhibits Rho-associated protein kinase and causes dephosphorylation of myosin light chain 2, which leads to dilatation of bile canaliculi of cultured hepatocytes (33) through dysregulation of cortical actin dynamics. When diameters of bile canaliculi increase, vesicles are formed at the apical membrane and released into the cytoplasm, followed by a decrease in canalicular diameter.…”